BOD analysis is presently carried out in accordance with Japanese Industrial Standard Method (JIS Industrial Wastewater Test Method K-0102-1972). Since it takes 5 days to analyze BOD, various attempts have been made for quick BOD analysis by utilizing microbe sensors. Among the microorganisms, Trichosporon cutaneum and activated sludge have been used as microbes immobilized on the microbe sensors (Japanese Patent Application KOKOKU No. 7258/1986; Suzuki S., ed. Biosensor pp135-136, 140-142 Kodansha Publication (1989)). In addition, an apparatus comprising a flow cell equipped with a microbe sensor has been known as a BOD analyzer (Japanese Patent Application KOKAI Nos. 47895/1978, 123851/1991).
The problem of the microbe sensor is that the BOD result of the microbe sensor has a low correlation to that of the JIS method. The problem is partly because of microorganisms used for microbe sensor. For example, Trichosporan cutaneum has a narrow assimilation spectrum on various organic substances, i.e. no response to disaccharides but specific high response to particular substances such as ethylalcohols. In addition, the microbe sensor has been impractical because the sensor has to be activated 1-3 days before BOD analysis in order to have microbes normally respond to samples. When activated sludge is used, activated sludge immobilized on a membrane has to be constantly controlled and the immobilization procedure is required every time the membrane is changed. BOD analysis is unable to carry out using any of the above microorganisms if high concentration of bactericidal substances such as arsenic contained in a sample.
In a BOD analyzer using a flow cell, bubbles remain in samples may affect analysis. In addition, it is necessary to have a more than 30-minute interval from one analysis to another to avoid an affect of a previous sample and it takes a long time to activate microbe membranes stored in a dried condition. Furthermore, maintenance and control of the BOD analyzer are cumbersome; various solutions have to be prepared in a large volume for analysis, which are easily decomposed and are frequently changed.
The present inventors have studied the problems described above and found that microorganisms belonging to the genus Klebsiella have the ability to assimilate a variety of organic substances and are activated in a short period of time by an activation procedure, the properties that are suitable for BOD analysis.
Although BOD analysis using a batch processing can be utilized for the present invention, the present inventors found that a micro-flow cell is useful for the BOD analyzer of the present invention because a micro-flow cell is advantageous to short residence time of samples, changes of solutions, and temperature control in a flow cell, the features that make the most of the properties of the microorganism and enable operators to carry out BOD analysis quickly and precisely.
In addition, the present inventors have developed a method of immobilizing microorganisms in a membrane, a BOD analyzer that shorted the time for analysis, a method of activating dried microbe membranes stored for a long period of time, and a method of maintaining the activation level of microorganisms in the microbe membrane using a minimum amount of solutions. These methods have overcome the disadvantages of the method of prior art, resulting in a practical BOD analyzer.